Electrophoresis is a technique in which molecules or other units move along a support medium under the influence of an electric field. Because different kinds of molecules and units behave differently, with migration rates depending on charge and frictional resistance, the technique is widely used for separation purposes. Commonly the support medium is a gel, and is used as in the form of a flat sheet; the mixture to be separated is placed at one end of the sheet; and a suitable electric field is applied along the sheet (from one end to the other) causing differential migration of the molecules or units in the mixture, resulting in separation.
Electrophoresis may be applied for many purposes, and is typically used for the separation of mixtures of biomolecules, that is molecules found in living organisms. These are typified by cell proteins and nucleic acids e.g. DNA (deoxyribonucleic acid) and fragments or denatured versions thereof, as well as many polysaccharides, polyphosphates, and proteoglycans.
It is naturally important that the gel itself should not be affected by the electric field
The most widely used anticonvective media for electrophoretic separations of bio-molecules are based on acrylamide polymers or agarose. Both of these are hydrophilic relatively stable polymers carrying practically no charge (polyacrylamide) or only a small number of charged groups (agarose) (see Johansson, B. G., Hjerten, S. (1974) Anal. Biochem. 59, 200-213.
The known acrylamide polymer gels are acrylamide crosslinked by N,N'-methylenebisacrylamide (Bis). They are referred to hereinafter as poly(acrylamide+Bis), and are excellent for the separation of molecules with molecular masses from 2 to 5 up to 1000 kDa, whether under denaturing or nondenaturing conditions (see Bothe, D., Simonis, M., and Dohren, H. V., (1985) Anal. Biochem. 151, 49-54; Campbell, W. P., Wrigley, C. M., and Margolis, J., (1983) Anal. Biochem. 129, 31-36; and Lambin, P., (1978) Anal. Biochem. 85, 114-125. Larger molecules or multimolecular complexes, however, cannot be satisfactorily resolved, due to the pronounced sieving effect-of the poly(acrylamide+Bis) gels even of the lowest workable concentration (approximately 3%). On the other hand, the more porous agarose gels are not suitable in some applications (see Bosisio, A. B., Loeherlein, C, Snyder, R. S., and Righetti, P. G., (1980) J. Chromatogr. 189, 317-330; and Righetti, P. G., (1986) Sci. Tools 33, 1-4, mostly because endo-osmosis, caused by the residual charged groups in the matrix, impairs resolution.
There have been numerous attempts to produce more porous polyacrylamide gels by increasing the concentration of Bis above 5% Rodbard, D., Levitov, C. and Chrambach, A. (1972), Sep. Sci. 7, 705-723; and Chrambach, A., and Rodbard, D., (1971) Science, 172, 440-451 or by using a new cross-linker Baumann, G., and Chrambach, A., (1976) Anal. Biochem. 70, 32-38. Moreover, composite polyacrylamide-agarose gels have also been tested as media for electrophoresis Bode, H. J. (1977) Anal. Biochem. 83, 204-210 and 364-371, and Pino, R. M. and Hart, T. K. (1984) Anal. Biochem. 139, 77-81.
In addition to electrophoretic analysis of large bio-molecules, highly porous matrices would be beneficial for other electrophoretic techniques where the extensive sieving of poly(acrylamide+Bis) gels represents a drawback. These include isoelectric focussing (IEF) in carrier ampholytes or in immobilised pH gradients (IPG) Righetti, P. G., (1983) in Isoelectric Focusing: Theory, Methodology, and Applications (Work, T. S. and Burdon, R. H. Eds.). Elsevier, Amsterdam and New York, as well as in titration curves Bosisio, A. B., Loeherlein, C, Snyder, R. S., and Righetti, P. G., (1980) J. Chromatogr. 189, 317-330. Furthermore, nonsieving media are required for stacking gels in multiphasic zone electrophoresis Jovin, T. M. (1973) Biochemistry, 12, 871-898, and Wyckoff, M., and Rodbard, D., and Chrambach, A., (1977), Anal. Biochem. 78, 459-482.
The present invention aims to provide novel polymers for use as gels suitable for use in electrophoresis.